Distinct Recognition of OX₁ and OX₂Receptors by Orexin Peptides

Abstract

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca 2ϩ responses (influx and release) in human OX 1 and OX 2 receptorexpressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca 2ϩ , suggesting similar activation of Ca 2ϩ influx as we have previously shown for orexin-A and OX 1 receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX 1 and OX 2 receptors, but the response mediated by the OX 2 receptor was more resistant to truncation than the response mediated by the OX 1 receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A 14 -33 . Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX 2 receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca 2ϩ dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX 2 receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10-to 100-fold lower affinity and potency for the OX 1 receptor.Recently, two novel hypothalamic peptides were isolated and subsequently named orexin-A and orexin-B (Sakurai et al., 1998) or hypocretin-1 and hypocretin-2 (de Lecea et al., 1998). Despite some initial confusion, orexin-A should now be considered identical to hypocretin-1 and orexin-B to hypocretin-2. Orexins act as agonists on two G-protein-coupled receptors called OX 1 and OX 2 receptors. Increased wakefulness and reduced sleep is a well demonstrated response to central administration of orexin, and disruption of central orexinergic signaling leads to the sleep disorder narcolepsy in animal models and probably also in man (reviewed in Beuckmann and Yanagisawa, 2002;Kukkonen et al., 2002;Sutcliffe and de Lecea, 2002). The other physiological roles for orexins may be regulation of energy homeostasis and stress response, probably both via central and peripheral mechanisms (reviewed in Willie et al., 2001;Beuckmann and Yanagisawa, 2002;Kirchgessner, 2002;Kukkonen et al., 2002;Smart and Jerman, 2002).The two orexin peptides, orexin-A and -B, are both products of the same precursor peptide, preproorexin, cleavage of which results in equimolar amounts of orexin-A and orexin-B. Orexin-A is composed of 33 amino acids and it contains two disulfide bridges, whereas orexin-B is a linear peptide of 28 residues (Sakurai et al., 1998). Although a product of a different part of the precursor peptide, orexin-B sho...

Antiallodynic Effects of Intrathecal Orexins in a Rat Model of Postoperative Pain

“…Nevertheless, it appears that the contribution of OX 1 receptors to the antiallodynic effect of intrathecal orexins is greater than that of OX 2 receptors. The finding that orexin A was more potent than orexin B may be attributed to the fact that orexin A displays 10-to 100-fold higher affinity for the OX 1 receptors than orexin B (Ammoun et al, 2003). P 2X Purinergic and Glycine, but not Opioid, Receptors are Involved in the Antiallodynic Effects of Orexins.…”

“…It might be attributed to the differences in the administration time of orexin B or in the pain animal models (Yamamoto et al, 2002). The mechanism(s) involved in chemically induced inflammatory pain and incision-induced mechanical allodynia might be different, such as the extent of sensory neuron sensitization and the pattern of spinal excitatory amino acid release (Vandermeulen and Brennan, 2000 Given that the antiallodynic effect of orexin B was not completely antagonized by SB-334867 and that orexin B has higher binding affinity for OX 2 than for OX 1 receptors (Ammoun et al, 2003), the possibility that the effect of orexin B is also mediated by OX 2 receptors remains to be elucidated. Nevertheless, it appears that the contribution of OX 1 receptors to the antiallodynic effect of intrathecal orexins is greater than that of OX 2 receptors.…”

The Orexin OX1Receptor Regulates Ca2+Entry via Diacylglycerol-Activated Channels in Differentiated Neuroblastoma Cells

“…This is frequently observed in heterologous expression systems with both OX 1 R and OX 2 R (Smart et al, 1999;Holmqvist et al, 2002;Ammoun et al, 2003) and is likely to be a consequence of activation of phospholipase C (PLC) and the generation of IP 3 . Some studies on native receptors in neurons have demonstrated sensitivity to inhibitors of phosphatidylinositol-specific PLC (Zhu et al, 2003;Muroya et al, 2004), suggesting that some store release may take place.…”

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

“…The localisation of the orexin proteins is widespread, but there are distinct differences between the two orexin subtypes and their receptors. While ORA is equipotent for OX1R and OX2R, ORB has an w10-fold higher affinity for the OX2 receptor subtype (Ammoun et al 2003). Additionally, the two receptor types appear to have differentially regulated b-arrestin-binding and internalisation profiles (Pfleger et al 2006).…”